Yersinia pestis, the causative agent of plague, must overcome the innate defenses of its host in order to establish an infection. In particular, Y. pests modulates its interaction with host phagocytic cells via a plasmid-encoded type III secretion system (T3SS) that functions to inject these cells with Yersinia outer proteins (Yops). A transposon site hybridization (TraSH)-based genome-wide screen was employed to identify additional genes required for the survival of Y. pestis KIM in the presence of phagocytic cells. This screen identified three chromosomal genes designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB) and C (ylrC) as being required for optimal survival in the presence of phagocytic cells. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. Strains carrying defined deletions of ylrA, ylrB, ylrC or ylrABC exhibited a defect in proliferation in the presence of macrophage like-RAW264.7 cells. C-terminal FLAG-tagged YlrA, YlrB and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium, indicating that the YlrA, YlrB and YlrC proteins are T3S substrates. Finally, expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins likely function as ant-host T3S effector proteins. The proposed studies will characterize the delivery, molecular mechanisms and roles in virulence of the ylrA, ylrB and ylrC gene products. Importantly, these proteins represent the first chromosome-encoded T3S effector proteins identified in Y. pestis.